Most cells in the human body are functionally polarized as reflected by different regions of the plasma membrane having distinct morphologies and protein compositions. The goal of the current research is to investigate mechanism that cells use to achieve this polarity. Specifically, this application focuses on the microfilament-based cytoskeleton of the apical aspect of polarized epithelial cells and how it contributes to the assembly, dynamics and composition of the abundant microvilli that characterize this domain. The membrane-cytoskeletal linker ezrin, the ezrin-binding scaffolding protein EBP50 and its ligand EPI64 are key proteins that control microvilli on the apical aspect of cells, and this proposal is to understand their individual and collective functions. Our experimental system utilizes the JEG3 cell line, derived from human placenta, as these cells express robust and dynamic microvilli. Ezrin is a very abundant, conformational regulated essential protein in microvilli, so we propose that its role in maintaining these structures is regulated in part by sampling the plasma membrane for appropriate binding partners. Therefore, the first aim seeks to establish a sensitive system to identify proteins that associate with the active conformation of ezrin, and then explore their role in microvilli maintenance. The second aim has two parts investigating EBP50, also essential for microvillar structure. Recent advances have shown that each of EBP50's two PDZ domains contribute to microvillar dynamics. First, proteins that bind EBP50's PDZ domains to regulate microvilli will be identified and characterized. Second, since microvilli are dynamic structures, and EBP50 is a highly dynamic component of them, we will use live cell imaging of wild type cells and those expressing characterized ezrin and EBP50 mutations to understand the relationship between the dynamics of components and the dynamics of a structure. The third aim concerns EPI64, an Arf6-GTP binding RabGAP protein that binds EBP50 and is now implicated in both an endocytic cycle and in regulating microvilli. The role of EPI64 and its interacting partners in endocytosis will be explored, as well as their impact on microvillus structure and dynamics. The fourth aim is to continue structural analyses of ezrin, EBP50 and EPI64 to gain insight into how these proteins function together. Overall, this proposal is to explore mechanisms involved in placing the right proteins at the right place and in the right abundance on the cell surface. These studies have relevance to many diseases. For example, the EBP50 ligands, CFTR and EGFR, both have to be present on the cell surface in the right abundance, and defects in their presence of over-abundance can cause cystic fibrosis or cancer, respectively.